To better understand the antigenic nature and the immune response to P. carinii, we have undertaken to characterize the major surface antigen of both rat and human P. carinii. It is necessary to use P. carinii from both sources because antigenically they are different and specifically the major surface antigen in rat and human are clearly different. We have previously purified the major surface glycoprotein (MSG) of both rat and human pneumocystis using HPLC. Subsequently, we identified a number of clones from a cDNA library of P. carinii that contain genes encoding for the MSG. The identity of these clones was confirmed by obtaining amino acid sequence information from purified MS(3. These clones are clearly related but not identical, very strongly suggesting that multiple genes encode the MSG. By pulse-field gel electrophoresis, these clones hybridize to all chromosomes. The predicted protein is high in cysteine and virtually all cysteine residues are conserved in all the clones. The predicted protein has a molecular weight of about 123,000. We have also generated peptides to poorly conserved regions for four of the clones that we have sequenced and have developed antibodies to these clones. In immunofluorescent and immunoblot studies we were able to show that two of these clones react preferentially with intact P. carinii. Over the past year we have attempted to characterize the genetic organization and regulation of this family of genes. Our evidence suggests that these genes are organized as tandem repeats, and that a single or limited number of sites regulate expression of the genes. Our collaborators at UCSF have demonstrated that a single upstream conserved sequence encoding a leader peptide is potentially expressed with each variable downstream MSG sequence, and we are attempting to demonstrate expression of this site using anti-peptide antibodies. Recently the human P. carinii MSG gene has been cloned, and a similar multi-gene family appears to encode this protein We are currently trying to clone and express some of these genes to allow studies of immune responses to P. carinii in humans. The goal of this study is to better understand the pathogenesis of P. carinii pneumonia with the hope that we can use this information to control or prevent this disease.